Vaccinia virus belongs to the genus Orthopoxvirus of the family of poxviruses. Certain strains of vaccinia virus have been used for many years as live vaccine to immunize against smallpox, for example the Elstree strain of the Lister Institute in the UK. Because of the complications which may derive from the vaccination (Schär, Zeitschr. für Präventivmedizin 18, 41–44 [1973]), and since the declaration in 1980 by the WHO that smallpox had been eradicated nowadays only people at high risk are vaccinated against smallpox.
Vaccinia viruses have also been used as vectors for production and delivery of foreign antigens (Smith et al., Biotechnology and Genetic Engineering Reviews 2, 383–407 [1984]). This entails DNA sequences (genes) which code for foreign antigens being introduced, with the aid of DNA recombination techniques, into the genome of the vaccinia viruses. If the gene is integrated at a site in the viral DNA which is non-essential for the life cycle of the virus, it is possible for the newly produced recombinant vaccinia virus to be infectious, that is to say able to infect foreign cells and thus to express the integrated DNA sequence (EP Patent Applications No. 83, 286 and No. 110, 385). The recombinant vaccinia viruses prepared in this way can be used, on the one hand, as live vaccines for the prophylaxis of infections, on the other hand, for the preparation of heterologous proteins in eukaryotic cells.
Vaccinia virus is amongst the most extensively evaluated live vectors and has particular features in support of its use as recombinant vaccine: It is highly stable, cheap to manufacture, easy to administer, and it can accommodate large amounts of foreign DNA. It has the advantage of inducing both antibody and cytotoxic responses, and allows presentation of antigens to the immune system in a more natural way, and it was successfully used as vector vaccine protecting against infectious diseases in a broad variety of animal models. Additionally, vaccinia vectors are extremely valuable research tools to analyze structure-function relationships of recombinant proteins, determine targets of humoral and cell-mediated immune responses, and investigate the type of immune defense needed to protect against a specific disease.
However, vaccinia virus is infectious for humans and its use as expression vector in the laboratory has been affected by safety concerns and regulations. Furthermore, possible future applications of recombinant vaccinia virus e.g. to generate recombinant proteins or recombinant viral particles for novel therapeutic or prophylactic approaches in humans, are hindered by the productive replication of the recombinant vaccinia vector. Most of the recombinant vaccinia viruses described in the literature are based on the Western Reserve (WR) strain of vaccinia virus. On the other hand, it is known that this strain is highly neurovirulent and is thus poorly suited for use in humans and animals (Morita et al., Vaccine 5, 65–70 [1987]).
Concerns with the safety of standard strains of VV have been addressed by the development of vaccinia vectors from highly attenuated virus strains which are characterized by their restricted replicative capacity in vitro and their avirulence in vivo. Strains of viruses specially cultured to avoid undesired side effects have been known for a long time. Thus, it has been possible, by long-term serial passages of the Ankara strain of vaccinia virus (CVA) on chicken embryo fibroblasts, to culture a modified vaccinia virus Ankara (MVA) (for review see Mayr, A., Hochstein-Mintzel, V. and Stickl, H. (1975) Infection 3, 6–14; Swiss Patent No. 568 392). The MVA virus was deposited in compliance with the requirements of the Budapest Treaty at CNCM (Institut Pasteur, Collectione Nationale de Cultures de Microorganisms, 25, rue de Docteur Roux, 75724 Paris Cedex 15) on Dec. 15, 1987 under Depositary No. I-721.
The MVA virus has been analysed to determine alterations in the genome relative to the wild type CVA strain. Six major deletions (deletion I, II, III, IV, V, and VI) have been identified (Meyer, H., Sutter, G. and Mayr A. (1991) J. Gen. Virol. 72, 1031–1038). This modified vaccinia virus Ankara has only low virulence, that is to say it is followed by no side effects when used for vaccination. Hence it is particularly suitable for the initial vaccination of immunocompromised subjects. The excellent properties of the MVA strain have been demonstrated in a number of clinical trials (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167, 375–390 [1987], Stickl et al., Dtsch. med. Wschr. 99, 2386–2392 [1974]).
Recently, a novel vaccinia vector system was established on the basis of the host range restricted and highly attenuated MVA virus, having foreign DNA sequences inserted at the site of deletion III within the MVA genome or within the TK gene (Sutter, G. and Moss, B. (1995) Dev. Biol. Stand. Basel, Karger 84, 195–200 and U.S. Pat. No. 5,185,146). Derived by longterm serial passage in chicken embryo fibroblasts (CEF), MVA can be propagated very efficiently in CEF, but it lost its capacity to grow productively in human and most other mammalian cells (Meyer, H., Sutter, G. and Mayr A. (1991) J. Gen. Virol. 72, 1031–1038 and Sutter et al., J. Virol., Vol. 68, No. 7, 4109–4116 (1994)). Viral replication in human cells is blocked late in infection preventing the assembly to mature infectious virions. Nevertheless, MVA is able to express viral and recombinant genes at high levels even in non-permissive cells and can serve as an efficient and exceptionally safe expression vector (Sutter, G. and Moss, B. (1992) Proc. Natl. Acad. Sci. USA 89, 10847–10851).
In animal models candidate vaccines on the basis of recombinant MVA have been found immunogenic and/or protective against a variety of infectious agents including influenzavirus, immunodeficiency viruses, and plasmodium parasites. Moreover, the potential usefulness of recombinant MVA for therapy of cancer has been established in several tumor model systems (Moss et al. 1996, Adv Exp Med Biol 397:7; Drexler et al. 1999, Cancer Res 59:4955). Interestingly, recombinant MVA vaccines induced equal or better immune responses to target antigens and were considerably less affected by preexisting vaccinia virus-specific immunity when compared to replication competent vaccina virus vectors (Ramirez et al. 2000, J. Virol 74:7651). Currently, MVA can be considered as the vaccinia virus strain of choice for vector development. Several recombinant MVA vaccines are already under clinical investigation in tumor immunotherapy and prophylaxis of human immunodeficiency virus infection.
The MVA genome contains several open reading frames (ORFs) coding for viral regulatory factors (Antoine et al. 1998, Virology 244:365). One of them is MVA ORF 050L (Antoine et al. 1998, Virology 244:365) also known as VV gene E3L (Goebel et al. 1990, Virology 179:247). The viral protein E3L is one of the key interferon (IFN) resistance factors encoded by VV (Smith et al. 1998, Sem. Virol. 8:409). It has been shown to bind dsRNA and inhibiting the activation of both PKR and 2′-5′ oligoadenylate synthetase (2-5A-S) (Chang et al. 1992, PNAS 89:4825; Rivas et al. 1998, Virology 243:406). E3L production has been described to be essential for VV replication in a range of mammalian host cells including human HeLa cells, but was found nonessential for virus propagation in CEF (Beattie et al. 1996, Virus Genes 12:89, Chang et al. 1995, J. Virol. 69:6605).
To further exploit the use of MVA, a novel way for the generation of recombinant MVA by introducing foreign genes by DNA recombination into the MVA strain of vaccinia virus has been sought. To generate recombinant MVA previously established strategies are based on the genomic co-insertion of selectable and non-selectable marker genes, e.g. the E. coli gpt and lacZ, or vaccinia virus K1L gene sequences (Sutter, G. and Moss, B. 1992, Proc. Natl. Acad. Sci. USA 89, 10847–10851; Staib, C. et al. 2000, Biotechniques, 28:1137–1148). The introduction of these heterologous markers into the MVA genome significantly improves the isolation of cloned recombinant viruses. However, during the cloning procedure there is need for supplementation of selective or chromogenic agents, such as mutagenic agent mycophenolic acid or X-Gal/DMFA, and/or the requirement for selective host cells. Furthermore, the maintenance of additional foreign gene sequences is not desireable for vector viruses to be used in clinical applications, and further genetical engineering of the viral genome is necessary to remove unwanted markers (Drexler et al. 1999, Cancer Res 59:4955; Staib, C. et al. 2000, Biotechniques, 28:1137–1148).